Cyclosporine Blocks Incorporation of HIV-1 Envelope Glycoprotein into Virions

ABSTRACT Cyclosporine (CsA) decreases HIV-1 infectivity by blocking HIV-1 capsid (CA) interaction with target cell cyclophilin A (CypA). Yet, HIV-1 virions produced in the presence of CsA also exhibit decreased infectivity that was previously shown to be independent of the well-characterized HIV-1 CA-CypA interaction. Here, we demonstrate that CsA decreases gp120 and gp41 incorporation into HIV-1 virions and that the fusion of these virions with susceptible target cells is impaired. This effect was not observed with HIV-1 virions pseudotyped with the vesicular stomatitis virus glycoprotein or with the amphotropic envelope protein of murine leukemia virus. It was independent of calcineurin signaling, the endoplasmic reticulum luminal protein cyclophilin B, and the long cytoplasmic tail of gp41. Thus, cyclosporine blocks HIV-1 infectivity via two independent mechanisms, the first involving HIV-1 CA in target cells and the second involving HIV-1 Env in producer cells.

Disruption of the Actin Cytoskeleton Can Complement the Ability of Nef To Enhance Human Immunodeficiency Virus Type 1 Infectivity

ABSTRACT The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.

Engraftment of NOD/SCID Mice with Human CD34+Cells Transduced by Concentrated Oncoretroviral Vector Particles Pseudotyped with the Feline Endogenous Retrovirus (RD114) Envelope Protein

ABSTRACT Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34+ (hCD34+) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34+ cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34+cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.